Dept. of Neurology-Neuroimmunology; Vall d'Hebron University Campus; Multiple Sclerosis Centre of Catalonia (Cemcat)

Author Of 3 Presentations

Disease Modifying Therapies – Mechanism of Action Poster Presentation

P0330 - Effects of cladribine on proliferation, survival and cytokine release of human astrocytes (ID 1581)

Speakers
Presentation Number
P0330
Presentation Topic
Disease Modifying Therapies – Mechanism of Action

Abstract

Background

Cladribine is a synthetic purine nucleoside analogue with immunosuppressive functions that has demonstrated beneficial effects in patients with relapsing-remitting multiple sclerosis (MS) and that may also regulate the immune function as an analogue of adenosine receptors. Although the effect of cladribine is well studied on immune cells, it remains unveiled how it affects the immune function of glial populations of the central nervous system.

Objectives

In the context of MS, we aimed to test the effect of cladribine on proliferation, survival and cytokine release of human astrocytes.

Methods

To assess the effect of cladribine on cell survival and proliferation, primary human astrocytes were cultured with cladribine at high concentrations (2µM, 0.2µM), at the mean estimated brain exposure of the drug (0.02µM) and at a low concentration (0.002µM) for 72h. The percentages of dead and proliferating cells were determined by flow cytometry. To assess the effect of cladribine on cytokine release, human astrocytes were stimulated for 6h with 20ng/ml IL1-β and TNF-α. The stimulus was withdrawn and cells were cultured for additional 18h. Cladribine was added for the whole 24h of culture. Supernatants were harvested to quantify IL1-β, IL6, TNF-α and GM-CSF release by Luminex. To assess the effect of cladribine independently of deoxycytidine kinase (DCK), deoxycytidine was also added to human astrocytes.

Results

Only high concentrations of cladribine induced death on human astrocytes (2µM: 35.89%±7.62 or 0.2µM: 7.27%±3.12 vs control: 3.17%1.84±; p<0.0001 and p=0.156, respectively) and inhibited their proliferative capacity (2µM: 0.96%±1.14 or 0.2µM: 14.06%±5.44 vs control: 33.06%±1.42; both p-values<0.0001). Additionally, the percentage of proliferating cells in the 2µM and 0.2µM conditions presented a limited capacity of proliferation (measured as the Relative Intensity of Proliferation Staining respect to basal; 2µM: 0.24±0.04 and 0.2µM: 0.55±0.22, both p-values<0.0001). When DCK activity was blocked by deoxycytidine, cell death and proliferation were reversed to control condition values. We are currently determining the effect of cladribine on pro-inflammatory cytokine release by human astrocytes.

Conclusions

The mean estimated brain exposure to cladribine does not influence cell survival or proliferation of human astrocytes neither in a DCK-dependent nor in a DCK-independent manner, suggesting that cladribine does not affect the normal astrocyte function in MS patients.

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Microbiome Poster Presentation

P0678 - Selected Clostridia strains increase responses related to interferon beta signaling and butyrate levels in experimental autoimmune encephalomyelitis (ID 1576)

Presentation Number
P0678
Presentation Topic
Microbiome

Abstract

Background

MS patients show a significant decrease in Clostridia clusters XIVa and IV in the gut microbiome. We have previously reported that a mixture of human gut-derived 17 Clostridia strains, which belong to Clostridia clusters XIVa, IV, and XVIII, improved the clinical outcome of experimental autoimmune encephalomyelitis (EAE) mice as a therapeutic approach. The clinical improvement was related to lower histopathological signs in the central nervous system (CNS) and to an enhanced immunoregulatory response of regulatory T (Treg) cells in the periphery.

Objectives

We aimed to study in depth the mechanism of action of the treatment with Clostridia strains in Experimental Autoimmune Encephalomyelitis

Methods

In two independent experiments, myelin oligodendrocyte glycoprotein (MOG)-immunized C57BL6/J mice were treated with Clostridia strains (n=15) or vehicle (n=15) via oral gavage from 13-14 days post-immunization (dpi) until the end of the experiment (28 dpi). At 28 dpi, spleens and spinal cords were collected to perform transcriptome studies and serum was collected to determine the short-chain fatty acid (SCFA) levels. In three independent experiments, MOG-immunized C57BL6/J mice were orally gavaged with butyrate (n=19) or vehicle (n=18) from 13-15 dpi until 28 dpi. Then, spinal cords were collected to perform histopathological studies.

Results

Therapeutic administration of Clostridia strains ameliorated EAE clinical course, as previously reported. Transcriptome studies revealed increased antiinflammatory responses related to interferon beta in the periphery and lower activation, differentiation, and proliferation of immune cells in the CNS. Higher levels of the immunomodulatory SCFA butyrate were detected in the serum of Clostridia-treated mice. Therefore, we studied the therapeutic effect of butyrate on EAE. We observed a slight therapeutic impact on EAE clinical course that was connected to a noticeable improve concerning axonal damage and a tendency to lower demyelination, inflammation, and astrogliosis in the CNS.

Conclusions

Clostridia strains perform their therapeutic effect on EAE enhancing the immunoregulatory response of Treg cells and antiinflammatory responses related to interferon beta signaling pathway in the periphery. The beneficial outcome exerted by the oral administration of the 17 Clostridia strains was not exclusively related to the production of the SCFA butyrate.

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Pathogenesis – Immunology Poster Presentation

P0972 - Impact of age on the immune system and the central nervous system in experimental autoimmune encephalomyelitis (ID 1598)

Speakers
Presentation Number
P0972
Presentation Topic
Pathogenesis – Immunology

Abstract

Background

The debut of multiple sclerosis (MS) at an older age associates with increased risk of presenting a primary progressive form, an earlier conversion to the secondary progressive form and a greater disability accumulation. These facts could be due to the impact of immunosenescence (ISC) in elderly MS patients.

Objectives

To study the impact of aging on clinical outcome, histopathology of the central nervous system (CNS) and peripheral immune system in experimental autoimmune encephalomyelitis (EAE).

Methods

8-week old and 40-week old C57BL/6JRccHsd female mice immunized with MOG35-55 were used. Histopathological and immunological studies were performed in non-immunized mice (basal) and in EAE mice 14 days post-immunization (dpi) and 28 dpi. Immunofluorescence staining was performed in spinal cords to evaluate T cell infiltration (CD3), demyelination (MBP), reactive astrogliosis (GFAP), reactive microglia (LEA) and axonal damage (SMI32). Immune cell subsets and intracellular cytokines were analyzed in splenocytes by flow cytometry. Polyclonal and MOG-specific capacity of proliferation were assessed in splenocytes. Differences between young and old mice in each EAE time point and along disease course were analyzed.

Results

Old mice (OM) showed a more severe EAE clinical outcome compared to young ones. Different patterns along EAE course were observed for inflammation, axonal damage and reactive microglia (increased at 28 dpi in OM) as well as for reactive astroglia (decreased at 14 dpi in OM) in the CNS. The adaptive immune cell subsets showed more age-related changes in OM, presenting a different pattern along EAE course: naïve CD8+ T cells (decreased basally and at 28 dpi), effector/effector memory CD4+PD1+ T cells (increased basally and at 14 dpi), regulatory CD39+ T cells (increased basally and at 14 dpi) and MHC-II+ B cells (increased basally and at 14 dpi). Regarding innate immune cells, immature NK cells increased and mature NK cells decreased along EAE course in OM, and NKT cells also presented a different pattern along EAE course (decreased basally and at 28 dpi in OM). Cytokine producing T cells were increased in OM. No differences were observed in splenocyte-proliferative capacity.

Conclusions

Aging has an impact on EAE outcome being more severe in old mice. Major changes take place in the CNS and in the adaptive immune cell subsets in the periphery. Altogether suggest that age modifies the immunopathogenic mechanisms of EAE.

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Presenter Of 1 Presentation

Microbiome Poster Presentation

P0678 - Selected Clostridia strains increase responses related to interferon beta signaling and butyrate levels in experimental autoimmune encephalomyelitis (ID 1576)

Presentation Number
P0678
Presentation Topic
Microbiome

Abstract

Background

MS patients show a significant decrease in Clostridia clusters XIVa and IV in the gut microbiome. We have previously reported that a mixture of human gut-derived 17 Clostridia strains, which belong to Clostridia clusters XIVa, IV, and XVIII, improved the clinical outcome of experimental autoimmune encephalomyelitis (EAE) mice as a therapeutic approach. The clinical improvement was related to lower histopathological signs in the central nervous system (CNS) and to an enhanced immunoregulatory response of regulatory T (Treg) cells in the periphery.

Objectives

We aimed to study in depth the mechanism of action of the treatment with Clostridia strains in Experimental Autoimmune Encephalomyelitis

Methods

In two independent experiments, myelin oligodendrocyte glycoprotein (MOG)-immunized C57BL6/J mice were treated with Clostridia strains (n=15) or vehicle (n=15) via oral gavage from 13-14 days post-immunization (dpi) until the end of the experiment (28 dpi). At 28 dpi, spleens and spinal cords were collected to perform transcriptome studies and serum was collected to determine the short-chain fatty acid (SCFA) levels. In three independent experiments, MOG-immunized C57BL6/J mice were orally gavaged with butyrate (n=19) or vehicle (n=18) from 13-15 dpi until 28 dpi. Then, spinal cords were collected to perform histopathological studies.

Results

Therapeutic administration of Clostridia strains ameliorated EAE clinical course, as previously reported. Transcriptome studies revealed increased antiinflammatory responses related to interferon beta in the periphery and lower activation, differentiation, and proliferation of immune cells in the CNS. Higher levels of the immunomodulatory SCFA butyrate were detected in the serum of Clostridia-treated mice. Therefore, we studied the therapeutic effect of butyrate on EAE. We observed a slight therapeutic impact on EAE clinical course that was connected to a noticeable improve concerning axonal damage and a tendency to lower demyelination, inflammation, and astrogliosis in the CNS.

Conclusions

Clostridia strains perform their therapeutic effect on EAE enhancing the immunoregulatory response of Treg cells and antiinflammatory responses related to interferon beta signaling pathway in the periphery. The beneficial outcome exerted by the oral administration of the 17 Clostridia strains was not exclusively related to the production of the SCFA butyrate.

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